In order to attempt to achieve the goals that we set ourselves we came up with three experiments to test our theories.
Experiment 1: Establishing Anchor Protein
1. Take a briobrick part (JO4450) and transform it into a chemically competent cell (Top 10)
2. Grow cells overnight then miniprep them to extract the transformed plasmid
3. Double digestion of plasmid to cut out the RFP gene, leaving only the plasmid backbone
4. PCR the full construct G-Block (containing Zif268 and Lpp_OmpA) and digest the product with the corresponding enzymes
5.
Golden gate assembly of plasmid backbones
and full construct.
6. Transform electrocompetent E.
coli cells with full plasmid
7. Grow cells
8. Miniprep full construct plasmid from modified cells
9. Digest plasmid with enzymes matching the cut sites around the Lpp_OmpA gene
10. PCR the rest of the anchor proteins from the G-blocks ordered (INP, PGSA, and BCLA)
11. Ligate the anchor proteins into digested full construct plasmid
12. Transform all plasmids into electrocompetent E.
coli - end product: four different types of cell, each expressing the full construct with slightly different transmembrane domains
13. Prepare transformed cells for microscopy (mount cells on glass slides)
14. Bind antibodies to FLAG tag - measure under fluorescent microscope
15. Determine whether the gene is being expressed and whether the expressed protein is being transported and is binding to the membrane of the E.
coli
16. Compare intensities - select anchor protein that fluoresces brightest, this anchor protein with be used for all subsequent experiments.
Experiment 2: Establishing Zinc Finger DNA Binding Proteins
1. Miniprep full construct plasmid from the first experiment
2. Digest plasmid with enzymes corresponding to the cut sites around the Zif268 gene
3. PCR zinc finger genes from the G-blocks ordered
4. Golden gate assembly of digested full construct plasmid and the three zinc finger PCR products
5. Transform electrocompetent E.
coli with each modified plasmid - end product: four different types of cells, each with the same anchor protein but different zinc finger DNA binding domains
6. Prepare the cells for microscopy by binding them to slides
7. Prepare four different sets of fluorescent oligos that each contain the target sequence for a zinc finger
8. Under a fluorescent microscope, wash the fixed cells with the fluorescent oligos, allow them to bind, then wash off the excess non-binded oligos
9. Measure fluorescence of the cells - cells that bind the fluorescent oligos on the surface will glow, allowing us to see that the zinc fingers are binding the target DNA sequence
10. Compare measurements to the same cell types washed with non-target sequence fluorescent DNA - if the cells have similar fluorescence it means that the zinc fingers are binding DNA indiscriminately, against what they were designed to do
11. Given that the zinc fingers bind DNA and discriminate between target and non-target DNA, select the cells that bind target DNA the best and grow these up
Experiment 3: Checking Colour
1. Make the selected E.
coli cells that express strong DNA binding zinc fingers from experiment 2 electro or chemically competent
2. Transform each E.
coli type with a unique colour fluorescent protein that expresses intracellularly - end product: three/four different E.
coli cell types, each expressing a different zinc finger and each glowing a different colour
3. Prepare slides by binding oligos with target DNA sequences to specific places on the slide to create a shape
4. Wash over the slide with complimentary E.
coli cells expressing the cognate zinc finger and allow the cells to bind, then wash the excess cells off the slide
5. Use a fluorescent microscope to determine whether the cells that stuck to the slide are bound in the predetermined pattern, indicating that they have bound themselves to the oligos printed on the slide
6. Repeat steps 3-5 using multiple different oligos and washing over with multiple cells to create a multi-coloured pattern
7. Repeat with more complex patterns
Techniques used
Fluorescence microscopy
In order to visualise our cells and determine whether the modified zinc finger protein was binding to the surface of our E. coli cell, we also included a FLAG tag domain in the protein.
This allowed us to use anti-FLAG antibodies to conduct an immuno-fluorescence test to see the location of the protein in the cells.
